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Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of ‘Iron addiction’, an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.

The use of CRISPR/Cas endonucleases has revolutionized gene editing techniques for research on Chlamydomonas reinhardtii. To better utilize the CRISPR/Cas system, it is essential to develop a more comprehensive understanding of the DNA repair pathways involved in genome editing. In this study, we have analyzed contributions from canonical KU80/KU70-dependent nonhomologous end-joining (cNHEJ) and DNA polymerase theta (POLQ)-mediated end joining on SpCas9-mediated untemplated mutagenesis and homology-directed repair (HDR)/gene inactivation in Chlamydomonas. Using CRISPR/SpCas9 technology, we generated DNA repair-defective mutants ku80, ku70, polQ for gene targeting experiments. Our results show that untemplated repair of SpCas9-induced double strand breaks results in mutation spectra consistent with an involvement of both KU80/KU70 and POLQ. In addition, the inactivation of POLQ was found to negatively affect HDR of the inactivated paromomycin-resistant mut-aphVIII gene when donor single-stranded oligos were used. Nevertheless, mut-aphVIII was still repaired by homologous recombination in these mutants. POLQ inactivation suppressed random integration of transgenes co-transformed with the donor ssDNA. KU80 deficiency did not affect these events but instead was surprisingly found to stimulate HDR/gene inactivation. Our data suggest that in Chlamydomonas, POLQ is the main contributor to CRISPR/Cas-induced HDR and random integration of transgenes, whereas KU80/KU70 potentially plays a secondary role. We expect our results will lead to improvement of genome editing in C. reinhardtii and can be used for future development of algal biotechnology.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1 - or BRCA2 -mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA -gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Polθ has been recently identified as a therapeutic target in cancer but specific inhibitors are currently unavailable. Here, the authors identify small molecule inhibitors of Polθ’s polymerase activity which elicit BRCA1/2 synthetic lethality, enhance the effect of PARP inhibitors and target PARP inhibitor resistance caused by 53BP1/Shieldin pathway defects.

Cellular pathways that repair chromosomal double-strand breaks (DSBs) have pivotal roles in cell growth, development and cancer. These DSB repair pathways have been the target of intensive investigation, but one pathway — alternative end joining (a-EJ) — has long resisted elucidation. In this Review, we highlight recent progress in our understanding of a-EJ, especially the assignment of DNA polymerase theta (Polθ) as the predominant mediator of a-EJ in most eukaryotes, and discuss a potential molecular mechanism by which Polθ-mediated end joining (TMEJ) occurs. We address possible cellular functions of TMEJ in resolving DSBs that are refractory to repair by non-homologous end joining (NHEJ), DSBs generated following replication fork collapse and DSBs present owing to stalling of repair by homologous recombination. We also discuss how these context-dependent cellular roles explain how TMEJ can both protect against and cause genome instability, and the emerging potential of Polθ as a therapeutic target in cancer.DNA polymerase theta (Polθ)-mediated end joining is a recently characterized DNA repair pathway that functions in various cellular contexts to repair DNA double-strand breaks that are not repaired by other pathways. Polθ-mediated end joining both helps maintain the genome and causes genome instability, and is an emerging therapeutic target in cancer.

DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

DNA double-strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle 1 . In interphase, DSBs are mainly repaired by non-homologous end joining and homologous recombination 2 . However, these pathways are completely inhibited in mitosis 3 – 5 , leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta 6 (Polθ) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Polθ function is activated in mitosis upon phosphorylation by Polo-like kinase 1 (PLK1). Phosphorylated Polθ is recruited by a direct interaction with the BRCA1 C-terminal domains of TOPBP1 to mitotic DSBs, where it mediates joining of broken DNA ends. Loss of Polθ leads to defective repair of mitotic DSBs, resulting in a loss of genome integrity. This is further exacerbated in cells that are deficient in homologous recombination, where loss of mitotic DSB repair by Polθ results in cell death. Our results identify mitotic DSB repair as the underlying cause of synthetic lethality between Polθ and homologous recombination. Together, our findings reveal the critical importance of mitotic DSB repair in the maintenance of genome integrity. In mitosis, genome integrity is maintained by DNA polymerase theta-dependent repair of DNA double-strand breaks, which is regulated by Polo-like kinase 1 activity.

DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ. DNA polymerase θ is a polymerase-helicase essential for microhomology-mediated end-joining (MMEJ) or alternative end-joining of DNA. Here the authors use biochemical and biophysical methods to reveal how full-length human DNA polymerase θ performs MMEJ at the molecular level.

Polymerase theta (Pol θ, gene name Polq ) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy. Polymerase theta is a widely conserved DNA polymerase that mediates Theta Mediated End Joining. Here authors present a synthetic lethal CRISPR screen to identify DDR gene mutations that induce cellular addiction to Pol theta.

Next-generation sequencing technology is used to show that the error-prone polymerase θ (Polθ) is needed to promote alternative non-homologous end joining at telomeres, and during chromosomal translocations, while counteracting homologous recombination; inhibition of Polθ represents a potential therapeutic strategy for tumours that have mutations in homology-directed repair genes. Polθ involved in alternative DNA repair The error-prone non-homologous end joining (NHEJ) DNA repair pathway is used as an alternative when the error-free homologous recombination pathway is compromised, or is the pathway of choice in some cellular contexts, such as in the immune system. After broken ends are paired via microhomology, NHEJ depends on DNA synthesis, but the identity of the polymerase involved was unclear. Two studies, from the laboratories of Agnel Sfeir and Alan D'Andrea, now implicate the mammalian POLQ gene, encoding the error-prone polymerase Polθ, in this process. Sfeir and colleagues show that upon telomere deprotection, Polθ is needed to prevent alternative end joining at telomeres, and chromosomal translations at non-telomeric sequences. D'Andrea and colleagues focus on the role of Polθ in cancer cells, and show that in a homologous-recombination-deficient background, the absence of Polθ results in a synthetic lethality, suggesting a possible therapeutic approach. The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions 1 , 2 , 3 . Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

In studies in mammalian cells, polymerase theta (Polθ, also known as POLQ) is identified as the polymerase responsible for non-homologous end joining DNA repair; this DNA repair pathway acts in many tumours when homologous recombination is inactivated and the identification of the polymerase responsible may aid the development of new therapeutic approaches. Polθ involved in alternative DNA repair The error-prone non-homologous end joining (NHEJ) DNA repair pathway is used as an alternative when the error-free homologous recombination pathway is compromised, or is the pathway of choice in some cellular contexts, such as in the immune system. After broken ends are paired via microhomology, NHEJ depends on DNA synthesis, but the identity of the polymerase involved was unclear. Two studies, from the laboratories of Agnel Sfeir and Alan D'Andrea, now implicate the mammalian POLQ gene, encoding the error-prone polymerase Polθ, in this process. Sfeir and colleagues show that upon telomere deprotection, Polθ is needed to prevent alternative end joining at telomeres, and chromosomal translations at non-telomeric sequences. D'Andrea and colleagues focus on the role of Polθ in cancer cells, and show that in a homologous-recombination-deficient background, the absence of Polθ results in a synthetic lethality, suggesting a possible therapeutic approach. Large-scale genomic studies have shown that half of epithelial ovarian cancers (EOCs) have alterations in genes regulating homologous recombination (HR) repair 1 . Loss of HR accounts for the genomic instability of EOCs and for their cellular hyper-dependence on alternative poly-ADP ribose polymerase (PARP)-mediated DNA repair mechanisms 2 , 3 , 4 , 5 . Previous studies have implicated the DNA polymerase θ (Polθ also known as POLQ, encoded by POLQ ) 6 in a pathway required for the repair of DNA double-strand breaks 7 , 8 , 9 , referred to as the error-prone microhomology-mediated end-joining (MMEJ) pathway 10 , 11 , 12 , 13 . Whether Polθ interacts with canonical DNA repair pathways to prevent genomic instability remains unknown. Here we report an inverse correlation between HR activity and Polθ expression in EOCs. Knockdown of Polθ in HR-proficient cells upregulates HR activity and RAD51 nucleofilament assembly, while knockdown of Polθ in HR-deficient EOCs enhances cell death. Consistent with these results, genetic inactivation of an HR gene ( Fancd2 ) and Polq in mice results in embryonic lethality. Moreover, Polθ contains RAD51 binding motifs and it blocks RAD51-mediated recombination. Our results reveal a synthetic lethal relationship between the HR pathway and Polθ-mediated repair in EOCs, and identify Polθ as a novel druggable target for cancer therapy.